Protein detection method

ABSTRACT

Described herein are systems and methods for detecting a target analyte in a sample with electrodes, comprising a linker and an antibody attached to the linker, and measuring an electrocatalytic signal changes generated by binding of an analyte in the sample to the antibody. Also disclosed herein are kits for electrochemical detection of protein analytes.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/354,356, filed Mar. 15, 2019, which is a continuation of U.S. patentapplication Ser. No. 15/684,218, filed Aug. 23, 2017, which is acontinuation of U.S. patent application Ser. No. 13/978,372, filed Dec.12, 2013, now U.S. Pat. No. 9,772,329, which is the U.S. national phaseof International Application No. PCT/US2012/020965, filed Jan. 11, 2012,which claims the benefit of priority to U.S. Provisional Application No.61/431,786, filed Jan. 11, 2011, each of which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

The development of platforms for the sensitive and straightforwardmeasurement of protein levels in clinical samples is an important goalthat will facilitate expanded use of protein biomarkers in diseasediagnosis. In order to provide useful information, detection schemesmust exhibit high levels of specificity, low limits of detection, androbust performance in biological fluids like blood and serum. Given theemergence of multi-protein signatures for cancer and other diseases,multiplexing is also a valuable feature. The inclusion of internal andexternal controls and calibrators—critical for the development ofaccurate diagnostic assays—also requires multiplexing.

A variety of high-performing protein detection platforms are underdevelopment, and many of the most specific and sensitive use micro- andnanomaterials in their sensing schemes. Barcoded nanoparticles, nanowiretransistors, enzyme-labeled beads, and microfluidic immunoarrays usedwith electrochemical readout all show promise for the development ofbiomarker analyzers. Challenges remain, however, pertaining to thedevelopment of simple analysis systems that are cost-effective androbust enough for clinical use.

SUMMARY OF THE INVENTION

Provided herein are detection systems for electrochemically detecting aprotein analyte. In one aspect, the detection systems comprise anelectrode comprising a linker on its surface, wherein the linker isattached to an antibody or fragment thereof capable of binding a proteinanalyte; and a redox reporter.

In some embodiments of the systems provided herein, the linker comprisesa functional group capable of direct or indirect coupling to theantibody or fragment thereof. In other embodiments, the linker comprisesa functional amine group. In yet other embodiments, the linker comprisesa functional carboxylic acid group. In further embodiments, the linkeris cystamine, cysteamine, mercapto propionic acid or 4-aminothiophenol.In yet further embodiments, the linker is attached to the antibody orfragment thereof via a second linker. In some instances, the secondlinker is glutaraldehyde or formaldehyde. In additional embodiments, thelinker is attached to multiple copies of an antibody or fragmentthereof.

In some embodiments of the systems provided herein, the antibody orfragment thereof is selected from the group consisting of polyclonalantiserum, polyclonal antibody, monoclonal antibody, Fab fragment, Fab′fragment, F(ab′)2 fragment, Fv fragment, single chain antibody, CDRpeptide and diabodies.

In some embodiments of the systems herein, the redox reporter is capableof generating an electrochemical signal with the electrode when apotential is applied. In other embodiments, the redox reporter generatesa faradaic current. In yet other embodiments, the redox reporter iscapable of interfacial electron transfer. In further embodiments, theredox reporter is ferricyanide/ferrocyanide or ferrocene. In yet furtherembodiments, the redox reporter ishexachloroiridate(IV)/hexachloroiridate(III).

In some embodiments of the systems provided herein, the electrode is anoble metal. In other embodiments, the electrode is carbon. In yet otherembodiments, the electrode is indium tin oxide. In further embodiments,the electrode is gold, palladium or platinum.

In some embodiments of the systems provided herein, the electrode is amicroelectrode. In certain embodiments of the systems provided herein,the electrode is a nanostructured microelectrode. In some embodiments,the electrode is less than about 500 microns. In other embodiments, theelectrode is less than about 250 microns. In still other embodiments,the electrode is less than about 100 microns. In yet other embodiments,the electrode is about 5 to about 50 microns. In further embodiments,the electrode is less than about 10 microns. In additional embodiments,the electrode is on a microfabricated chip. In further embodiments arepresent a plurality of electrodes arrayed on a substrate.

In some embodiments of the systems provided herein, the protein analyteis a biomarker for a disease, disorder or condition. In some instancesthe biomarker is a cancer biomarker. In certain instances, the biomarkeris selected from the group consisting of BRCA1, BRCA1, Her2/neu,alpha-feto protein, beta-2 microglobulin, bladder tumor antigen, cancerantigen 15-3, cancer antigen 19-9, human chorionic gonadotropin, cancerantigen 72-4, cancer antigen 125 (CA-125), calcitonin, carcino-embryonicantigen, EGFR, Estrogen receptors, Progesterone receptors, Monoclonalimmunoglobulins, neuron-specific enolase, NMP22, thyroglobulin,progesterone receptors, prostate specific antigen (PSA),prostate-specific membrane antigen, prostatic acid phosphatase, S-100,and TA-90, or a portion, variation or fragment thereof. In furtherinstances, the biomarker is a biomarker for Staphylococcus orStreptococcus bacterial infections.

Also provided herein are methods for electrochemical detection of aprotein analyte. In one aspect, the methods comprise contacting anelectrode comprising a linker on its surface, wherein the linker isattached to an antibody or fragment thereof capable of binding a proteinanalyte with a sample and a redox reporter; measuring an electrochemicalsignal generated by the antibody-labeled electrode and the redoxreporter when a potential is applied; and comparing the electrochemicalsignal to a signal of a control sample comprising no protein analyte;wherein a change of the signal detected relative to a signal of acontrol sample comprising no protein analyte is indicative of thepresence of the protein analyte in the sample.

In some embodiments of the methods provided herein, the linker comprisesa functional group capable of direct or indirect coupling to theantibody or fragment thereof. In other embodiments, the linker comprisesa functional amine group. In yet other embodiments, the linker comprisesa functional carboxylic acid group. In further embodiments, the linkeris cystamine, cysteamine, mercapto propionic acid or 4-aminothiophenol.In yet further embodiments, the linker is attached to the antibody orfragment thereof via a second linker. In some instances, the secondlinker is glutaraldehyde or formaldehyde. In additional embodiments, thelinker is attached with multiple copies of an antibody or fragmentthereof.

In some embodiments of the methods provided herein, the antibody orfragment thereof is selected from the group consisting of polyclonalantiserum, polyclonal antibody, monoclonal antibody, Fab fragment, Fab′fragment, F(ab′)2 fragment, Fv fragment, single chain antibody, CDRpeptide and diabodies.

In some embodiments of the systems herein, the redox reporter generatesa faradaic current. In other embodiments, the redox reporter is capableof interfacial electron transfer. In further embodiments, the redoxreporter is ferricyanide/ferrocyanide or ferrocene. In yet furtherembodiments, the redox reporter ishexachloroiridate(IV)/hexachloroiridate(III).

In some embodiments of the methods provided herein, the electrode is anoble metal. In other embodiments, the electrode is carbon. In yet otherembodiments, the electrode is indium tin oxide. In further embodiments,the electrode is gold, palladium or platinum.

In certain embodiments of the methods provided herein, the electrode isa nanostructured microelectrode. In other embodiments, the electrode isless than about 100 microns. In yet other embodiments, the electrode isabout 5 to about 50 microns. In further embodiments, the electrode isless than about 10 microns. In additional embodiments, the electrode ison a microfabricated chip.

In some embodiments of the methods provided herein, the protein analyteis a biomarker for a disease, disorder or condition. In some instancesthe biomarker is a cancer biomarker. In certain instances, the thebiomarker is selected from the group consisting of BRCA1, BRCA1,Her2/neu, alpha-feto protein, beta-2 microglobulin, bladder tumorantigen, cancer antigen 15-3, cancer antigen 19-9, human chorionicgonadotropin, cancer antigen 72-4, cancer antigen 125 (CA-125),calcitonin, carcino-embryonic antigen, EGFR, Estrogen receptors,Progesterone receptors, Monoclonal immunoglobulins, neuron-specificenolase, NMP22, thyroglobulin, progesterone receptors, prostate specificantigen (PSA), prostate-specific membrane antigen, prostatic acidphosphatase, S-100, and TA-90, or a portion, variation or fragmentthereof. In further instances, the biomarker is a biomarker forStaphylococcus or Streptococcus bacterial infections.

Also provided herein are methods for multiplexed electrochemicaldetection of a plurality of protein analytes. In one aspect, the methodscomprise contacting a first electrode comprising a linker on itssurface, wherein the linker is attached to a first antibody or fragmentthereof capable of binding a protein analyte with a sample and a redoxreporter; measuring a first electrochemical signal generated by thefirst antibody-labeled electrode and the redox reporter when a potentialis applied; contacting a second electrode comprising a linker on itssurface, wherein the linker is attached to a second antibody or fragmentthereof capable of binding a protein analyte with a sample and a redoxreporter; measuring a second electrochemical signal generated by thesecond antibody-labeled electrode and the redox reporter when apotential is applied; and comparing the first and second electrochemicalsignals to respective signals generated by the first and secondantibody-labeled electrode in a control sample comprising no proteinanalyte; wherein a change of the first and second electrochemicalsignals detected relative to the respective signals of a control samplecomprising no protein analyte is indicative of the presence of theprotein analyte in the sample.

In some embodiments of the methods provided herein, the first and secondelectrode are both on a microfabricated chip. In other embodiments, thefirst and second electrode are on different microfabricated chips.

In some embodiments of the methods provided herein, the secondantibody-labeled electrode is a reference control to the firstantibody-labeled electrode. In other embodiments, the secondantibody-labeled electrode detects abundant serum protein.

Also provided herein are methods for monitoring progression or responsein a subject having a cancer. In one aspect, the methods compriseobtaining a biological sample from the subject; contacting an electrodecomprising a linker on its surface, wherein the linker is attached to anantibody or fragment thereof capable of binding a protein analyte withthe sample and a redox reporter wherein the antibody or fragment thereofbinds to a protein analyte; measuring an electrochemical signalgenerated by the antibody-labeled electrode and the redox reporter whena potential is applied; and comparing the electrochemical signal to asignal of a control sample comprising no protein analyte; wherein achange of the signal detected relative to a signal of a control samplecomprising no protein analyte is indicative of the presence of theprotein analyte in the sample.

In some embodiments of the methods provided herein, the protein analyteis a biomarker for a disease, disorder or condition. In some instancesthe biomarker is a cancer biomarker. In certain instances, the thebiomarker is selected from the group consisting of BRCA1, BRCA1,Her2/neu, alpha-feto protein, beta-2 microglobulin, bladder tumorantigen, cancer antigen 15-3, cancer antigen 19-9, human chorionicgonadotropin, cancer antigen 72-4, cancer antigen 125 (CA-125),calcitonin, carcino-embryonic antigen, EGFR, Estrogen receptors,Progesterone receptors, Monoclonal immunoglobulins, neuron-specificenolase, NMP22, thyroglobulin, progesterone receptors, prostate specificantigen (PSA), prostate-specific membrane antigen, prostatic acidphosphatase, S-100, and TA-90, or a portion, variation or fragmentthereof. In further instances, the biomarker is a biomarker forStaphylococcus or Streptococcus bacterial infections.

Also provided herein are kits for electrochemical detection of a proteinanalyte. In one aspect, the kits comprise an electrode comprising alinker on its surface, wherein the linker is attached to an antibody orfragment thereof capable of binding a protein analyte; and a redoxreporter capable of generating an electrochemical signal with theelectrode when a potential is applied.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A. Photograph (left) of a multiplexed sensor chip showingmicrofabricated chip featuring 5 μm openings for the electrochemicaldeposition of electrodes and an illustration (middle) of aperture. Agold (Au) pattern is deposited on a silicon wafer using conventionalphotolithography and is then covered with a layer of SiO₂; 5 μm openingsare then etched through this top layer to expose a circular section ofAu. Schematic illustration of the generation of Au electrodes by Auelectrodeposition (right).

FIG. 1B. Schematic of electrode functionalization; (left) a linker ofcystamine is formed on Au structure; (middle) reaction with abifunctional linker glutaraldehyde to introduce aldehyde groups at thesensor surface; (right) addition of the anti CA-125 antibody or antiHuman Serum Albumin (HSA) antibody to prepare antibody-modifiedelectrode sensors.

FIG. 1C. Schematic view of electrochemical detection for CA-125 antigen.The antigen-antibody binding hinders the interfacial electron transferreaction of [Fe(CN₆]_(3-/4-).

FIG. 1D. Differential pulse voltammetry (DPV) showing the signaldecrease observed after incubation of the CA-125 (10 U/ml in serum) for40 min.

FIGS. 2A-2G. SEM images and characteristic cyclic voltammograms of threedifferently-sized AU electrode sensors. SEM images of (a) 100 micronsensor. This structure was fabricated using DC potential amperometry atan applied potential of 0 mV for 200 s, (c) 30 micron sensor. Thisstructure was fabricated using DC potential amperometry at an appliedpotential of 150 mV for 200 s, and (e) 8 micron sensor. This structurewas fabricated using chronopotentiometry at an applied current of 30 nAfor 50 s. Characteristic cyclic voltammograms of the three sensors wereobtained in a 10 mM phosphate buffer solution containing 2.5 mM[Fe(CN)₆]_(3-/4-) and 0.1 M. KCl at a scan rate of 100 mV/s at sensorswith size (b) 100 micron, (d) 30 micron, and (f) 8 micron sensors. Insetof FIG. 2(f) shows the magnified view of the cyclic voltammogram. (g)Capacitive current of three sensors reflecting surface area for eachsensor size. Cyclic voltammograms were obtained in a 10 mM phosphatebuffer solution containing 0.1 M. KCl at a scan rate of 100 mV/s atsensors with size (outer curve) 100 micron, (middle curve) 30 micron,and (inner curve) 8 micron.

FIG. 3A-3C. Comparison of the sensitivities and detection limits of theimmunosensors generated at three differently-sized Au structures. AA(1%)with concentration of CA-125 in PBS were obtained with (a) 100 micronsensor, (b) 30 micron sensor, and (c) 8 micron sensor.

FIG. 4A-4B. Change of current of the sensors with size (a) 100 micronand (b) 8 micron before (grey bar) and after (black bar) incubation withdifferent concentrations of CA-125 in PBS.

FIG. 5A-5B. Detection of CA-125 in serum and whole blood. (a)Simultaneous detection of CA-125 and HSA in spiked serum samples. Samplecontained undiluted serum. Data obtained with serum only and serumspiked with different concentrations of CA-125. Grey bars indicate thedata obtained with anti CA-125 antibody-modified immunosensors and blackbars represent that for anti HSA antibody-modified immunosensors. Sensorsize was 8 micron. (b) Detection of CA-125 in whole blood. Samplescontained undiluted, unprocessed blood and the concentrations of CA-125indicated.

DETAILED DESCRIPTION OF THE INVENTION I. Electrochemical DetectionSystems and Methods

Provided herein are systems and methods for electrochemically detectinga target analyte in a sample. The presence of an analyte is detected bya change in an electrocatalytic signal. The use of such an electricalreadout provides a method which is inexpensive, extremely sensitive,easy to miniaturize and easy to automate.

In one aspect of the detection systems and methods described herein, anelectrode is provided wherein the electrode comprises a linker andwherein the linker is attached to an antibody or fragment thereof. Theantibody or fragment thereof is capable of binding to a target analytesuch as a protein. The electrode is in the presence of a redox reporter.

Redox reporters suitable for use in the systems and methods describedherein are capable of generating an electrical signal (e.g., faradaiccurrent) with the electrode when a potential is applied. Any redoxreporter that generates a faradaic current or is capable of interfacialelectron transfer with the electrode can be used. Non-limiting redoxreporters, include but are not limited to small redox-active groups suchas ferricyanide/ferrocyanide, ferrocene andhexachloroiridate(IV)/hexachloroiridate(III). The detection systemsutilize redox reporters to generate baseline electrical signals with theelectrode. When a target analyte is present that binds to the antibodyor fragment thereof, the electrical signal is attenuated. It iscontemplated that attenuation of the signal is due to the target analyteblocking the redox reporter from effectively accessing the surface ofthe electrode. In other words, the antibody-analyte binding hindersinterfacial electron transfer. See, by way of example only, FIGS. 1c and1 d.

In one aspect, the signal changes corresponding to target analytebinding to the antibody are calculated as a percentage change infaradaic current:

ΔI%={(mean I ₀)−(mean I _(c))}/mean I= ₀×100

where mean I₀=mean current at zero target concentration, mean I_(c)=meancurrent at any concentration of target). In certain embodiments, thesignal change is at least about 10%, at least about 15%, about 25%,about 30%, about 40%, about 50%, about 65%, about 75%, about 85%, about90%, about 95%, about more than 100%, about twofold, about ten fold,about fifty fold, or greater. In certain instances, a change of thesignal indicates that the analyte is bound to the antibody. With thechange in the faradaic current, the detection systems and methodsdescribed herein are used in one aspect to determine the presence of atarget analyte.

In another aspect, the detection systems and methods described hereinare used to determine the concentration of a target analyte in a sample.In some embodiments, this is achieved via calibration of the detectionsystem with known concentration standards of the target analyte. Forexample, a number of positive control samples, each with specificconcentrations of analyte, are used to determine the percentage changein faradaic current for determination of an unknown quantity of ananalyte in a test sample. The detection ranges for the detection systemsand methods are dependent on the antibody, analyte and there bindingcapabilities as well as the redox reporter used. In some embodiments,the detection systems and methods described herein detect concentrationsof analyte at about 500 femtomolar (fM) or about 100 pg/mL or lower.

In another aspect, the detection systems and methods described hereinare multiplexed for detecting and/or determining the concentration of aplurality of target analytes. In some embodiments, multiplexed systemsand methods comprise at least two electrodes, each comprising a linkerwith different antibodies attached to each linker. In certain instances,two, three, four, five, six, seven, eight, nine or ten electrodes, eachcomprising a linker with different antibodies attached to each linkerare employed in a multiplexed system. In some embodiments, at leastthree, at least five, at least ten, at least fifteen, at least twenty,at least thirty, at least forty, or at least fifty or more electrodesare employed in a multiplexed system, each comprising a linker withdifferent antibodies attached to each linker at each electrode.Alternatively, more than one electrode may contain the same antibody orantibody class; for example, duplicates of four electrodes, each groupcontaining one of three separate antibodies, may be used in atwelve-electrode multiplexed system. Furthermore, an electrode maycontain more than one antibody or antibody class; for example, on anindividual electrode, more than one antibody, each recognizing aspecific region of a protein or analyte, may be combined. In someinstances, detection will occur only if a protein or analyte binds toall antibodies bound to the electrode. In some instances, detection willoccur if a protein or analyte binds to at least one of the antibodygroups bound to the electrode.

Multiplexing allows for a large variety of analytes to be detectedsimultaneously, thus creating an “analyte panel”. Exemplary analytepanels can contain analytes related to a disorder, disease or condition,e.g., related biomarkers for a certain cancer. Multiplexing also allowsfor greater sensitivity for an analyte such as different antibodies thatbind to the same target analyte via the same or different epitopes. Theuse of different antibodies, such as, for example, the use of apolyclonal and a monoclonal antibody that target the same analyte,allows the detection system to be more robust and sensitive than asingle-plex system that uses only one type of antibody to detect ananalyte. Multiplexing further allows internal calibration of the systemto reduce false positives and negatives. For example, analysis of atarget analyte can be performed in parallel with an analyte known to bestable such as an abundant serum protein.

In another aspect, the detection systems and methods described hereinare employed to detect or diagnose a disorder, disease or condition orto monitor progression or response of a disorder, disease or condition.In some embodiments, a sample is obtained from a patient or subject andthe detection systems and methods are used to detect the presence ofand/or the concentration of a target analyte associated with thedisorder, disease or condition. Exemplary disorders, diseases orconditions include cancers (e.g., breast, ovarian, prostate, pancreatic,colorectal, bladder and the like), infectious diseases (e.g.,Staphylococcus or Streptococcus bacterial infections, MRSA, VISA, viralinfections, fungal infections and the like), autoimmune diseases (e.g.,Graves' disease, Lupus, arthritis, Goodpasture's syndrome and the like),metabolic disease and disorders (e.g., metabolic syndrome, insulinresistance, diabetes type I and II, Crohn's disease, irritable bowelsyndrome and the like), HIV/AIDS, genetic diseases, and conditionsassociated with therapeutic drugs or toxicologic materials. Severity orstages of a disorder, disease or condition are determined in someembodiments by detecting the concentration of the target analyte wheredifferent concentrations indicate severity or stage. Likewise, in otherembodiments, progression or response of a disorder, disease or conditionare determined by detecting the concentration of the target analyteacross various time points. Therapeutic effective of a pharmacologicaltreatment, therapy or regimen can, in some embodiments, also bedetermined by detecting the concentration of the target analyte acrossvarious time points.

II. Electrodes

Electrodes for the detection systems and methods described herein areany electrically conductive materials with properties allowing linkerson the electrode's surfaces. Electrodes have the capability to transferelectrons to or from a redox reporter and are generally connected to anelectronic control and detection device. In general, noble metals, suchas, Ag, Au, Ir, Os, Pd, Pt, Rh, Ru and others in their family aresuitable materials for electrodes. Noble metals have favorableproperties including stability and resistance to oxidation, may bemanipulated in various methods such as electrodeposition, and bind tothiols and disulfide containing molecules thereby allowing attachment ofsaid molecules. Other materials can also be used, such asnitrogen-containing conductive compounds (e.g., WN, TiN, TaN) orsilicon/silica-based materials, such as silane or siloxane. In certainembodiments, the electrode is gold, palladium or platinum. In otherembodiments, the electrode is carbon. In further embodiments, theelectrode is indium tin oxide.

In some embodiments, the electrode is a microelectrode. In otherembodiments, the microelectrode is a nanostructured microelectrode(“NME”). NMEs are microelectrodes that feature nanostructured surfaces.Surface nanotexturing or nanostructures provide the electrode with anincreased surface area, allowing for greater sensitivity, particularlyin biosensing applications. Manufacturing of NMEs can be performed viaelectrodeposition. By varying parameters such as deposition time,deposition potential, supporting electrolyte type and metal ion sources,NMEs of a variety of sizes, morphologies and compositions may begenerated. In certain instances, NMEs have a dendritic structure.Complexity of the dendritic structure is achieved by the varying theaforementioned electrodeposition parameters. Exemplary NMEs for use inthe systems and methods described herein are described in InternationalPat. Appl. Ser. No. PCT/CA2009/001212 (published as WO/2010/025547)which is incorporated by reference in its entirety.

Other electrode structures can also be used in the detection systems andmethods described herein, including, planar surfaces, wires, tubes,cones and particles. Commercially available macro- and micro-electrodesare also suitable for the embodiments described herein.

Electrodes are sized, for example, from between about 0.0001 to about5000 microns in length or diameter; between about 0.0001 to about 2000microns in length or diameter; from between about 0.001 to about 250microns; from between about 0.01 to about 200 microns; from betweenabout 0.1 to about 100 microns; from between about 1 to about 50microns; from between about 10 to about 30 microns in length, or belowabout 10 microns in length or diameter. In certain embodiments,electrodes are sized at about 100 microns, about 30 microns, about 10microns or about 5 microns in length or diameter. In furtherembodiments, electrodes are sized at about 8 microns.

In some embodiments, the detection systems and methods described herein,comprise one electrode for detection. In other embodiments, multipleelectrodes are used. Use of multiple electrodes can be used in parallelto detect a target analyte via one antibody type attached to eachelectrode, in some embodiments. Alternatively, in other embodiments,multiple electrodes are used for multiplexing as described previously.Multiple electrodes can be configured in high or low density arrays. Anexemplary 8 electrode array on a microfabricated chip for multiplexinguse is depicted in FIG. 1 a.

In further embodiments, an electrode is located upon a substrate. Thesubstrate can comprise a wide range of material, either biological,nonbiological, organic, inorganic, or a combination of any of these. Forexample, the substrate may be a polymerized Langmuir Blodgett film,functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,cross-linked polystyrene, polyacrylic, polylactic acid, polyglycolicacid, poly(lactide coglycolide), polyanhydrides, poly(methylmethacrylate), poly(ethylene-co-vinyl acetate), polysiloxanes, polymericsilica, latexes, dextran polymers, epoxies, polycarbonates, orcombinations thereof.

Substrates can be planar crystalline substrates such as silica basedsubstrates (e.g. glass, quartz, or the like), or crystalline substratesused in, e.g., the semiconductor and microprocessor industries, such assilicon, gallium arsenide, indium doped GaN and the like. Silicaaerogels can also be used as substrates, and can be prepared by anyknown methods. Aerogel substrates may be used as free standingsubstrates or as a surface coating for another substrate material.

The substrate can take any form and typically is a plate, slide, bead,pellet, disk, particle, microparticle, nanoparticle, strand,precipitate, optionally porous gel, sheets, tube, sphere, container,capillary, pad, slice, film, chip, multiwell plate or dish, opticalfiber, etc. The substrate can be any form that is rigid or semi-rigid.The substrate may contain raised or depressed regions on which an assaycomponent is located. The surface of the substrate can be etched usingwell known techniques to provide for desired surface features, forexample trenches, v-grooves, mesa structures, or the like. The substratecan take the form of a photodiode, an optoelectronic sensor such as anoptoelectronic semiconductor chip or optoelectronic thin-filmsemiconductor, or a biochip. The location(s) of electrode(s) on thesubstrate can be addressable; this can be done in highly dense formats,and the location(s) can be microaddressable or nanoaddressable. In someembodiments, the electrode(s) is on a microfabricated chip.

Surfaces on the substrate can be composed of the same material as thesubstrate or can be made from a different material, and can be coupledto the substrate by chemical or physical means. Such coupled surfacesmay be composed of any of a wide variety of materials, for example,polymers, plastics, resins, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, membranes, or any of theabove-listed substrate materials.

The substrate and/or its surface is generally resistant to, or istreated to resist, the conditions to which it is to be exposed in use,and can be optionally treated to remove any resistant material afterexposure to such conditions.

III. Linkers

In one aspect, the electrode comprises a linker on the surface of theelectrode. Linkers, in some embodiments, can be formed when linkermolecules absorb and are organized into a molecular layer on a surface.Linkers suitable for use with the electrodes disclosed herein have a“head group” that strongly chemisorbs with metals (e.g., thiols anddisulfides) and a tail with a functional group (e.g., —OH, —NH₂, —COOH,—CO, —OCH₃, —NHNH₂, -biotin, —NHS (amine-reactive N-hydroxysuccimide)).Examples of linkers include single chain or branched chain alkylthiolswith a functional group. Other linker molecules include aromatic thiolssuch as thiophenol with a functional group. Suitable linker moleculesinclude any molecule with a functional group that can directly orindirectly link to an antibody. Exemplary linker molecules include, butare not limited to, cystamine, cysteamine, mercapto propionic acid or4-aminothiophenol. In some embodiments, the linker is cystamine.

Linkers arc formed on the electrode surface when the electrode isimmersed a solution of the linker molecule. Typical concentrationscontain about 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, about 2 mM, about5 mM, about 10 mM, about 20 mM or about 50 mM or more of the linkermolecule in an aqueous or ethanolic solution. The immersion is over aperiod of time ranging from a few hours to days. In some embodiments,the immersion is about 4 hours, about 8 hours, about 16 hours, about 24hours, about 2 days, about 5 days or about 7 days. In some embodiments,the immersion is at room temperature. In other embodiments, theimmersion is above room temperature. In further embodiments, theimmersion is lower than room temperature.

Linkers can be attached directly or indirectly to an antibody by anyknown method. Direct attachment can, in some embodiments, be achievedthrough the functional groups of the linker molecules such as —COfunctional groups that can react and attach to antibodies.Alternatively, a second linker or spacer can be conjugated onto thefunctional group by which the second linker or spacer can thereby attachto the antibody, in other embodiments. For example, linker moleculeswith —NH functional groups can react with a linker such asgluteraldehyde or formaldehyde which in turn can attach to antibodies.In further embodiments, the antibody can be derivatized so as tointeract with functional group. For example, an avidin-labeled antibodycan attach to a biotin functional group of a linker. These and otherexamples direct and indirect linkage are within the scope of theembodiments described herein.

IV. Antibodies

In one aspect, an antibody is used for determining the amount and/orconcentration of a target analyte. Antibodies belong to a family ofplasma proteins called immunoglobulins, whose basic building block, theimmunoglobulin fold or domain, is used in various forms in manymolecules of the immune system and other biological recognition systems.A typical immunoglobulin has four polypeptide chains, containing anantigen binding region known as a variable region and a non-varyingregion known as the constant region. An antibody that is suitable foruse in the present embodiments herein can be in any of a variety offorms, including, sera, a whole immunoglobulin, an antibody fragmentsuch as Fv, Fab, and similar fragments, a single chain antibody whichincludes the variable domain complementarity determining regions (CDR),and the like forms, all of which fall under the broad term “antibody”,as used herein. The present embodiments herein contemplate the use ofany specificity of an antibody, polyclonal or monoclonal, and is notlimited to antibodies that recognize and immunoreact with a specificantigen In some embodiments herein, in the context of both thetherapeutic and screening methods, an antibody or fragment thereof isused that is immunospecific for a target analyte.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. The monoclonal antibodies herein specifically include“chimeric” antibodies (immunoglobulins) in which a portion of the heavyand/or light chain is identical with or homologous to correspondingsequences in antibodies derived from a particular species or belongingto a particular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity. See, e.g., U.S. Pat. No.4,816,567; Morrison et al. Proc. Natl. Acad Sci. 81, 6851 6855 (1984).

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)2 and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)2 fragment thathas two antigen binding fragments which are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)2 fragments.

Antibody fragments retain some ability to selectively bind with itsantigen or receptor and are defined as follows:

(1) Fab is the fragment that contains a monovalent antigen-bindingfragment of an antibody molecule. A Fab fragment can be produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain.

(2) Fab′ is the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain. Two Fab′ fragmentsare obtained per antibody molecule. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxyl terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region.

(3) F(ab′)2 is the fragment of an antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction. F(ab′)2 is a dimer of two Fab′ fragments held together by twodisulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in a tight, non-covalentassociation (V_(H) V_(L) dimer). It is in this configuration that thethree CDRs of each variable domain interact to define an antigen bindingsite on the surface of the V_(H) V_(L) dimer. Collectively, the six CDRsconfer antigen binding specificity to the antibody. However, even asingle variable domain (or half of an Fv comprising only three CDRsspecific for an antigen) has the ability to recognize and bind antigen,although at a lower affinity than the entire binding site.

(5) Single chain antibody (“SCA”), defined as a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule. Such single chain antibodiesare also referred to as “single-chain Fv” or “sFv” antibody fragments.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains that enables the sFv to form the desiredstructure for antigen binding. For a review of sFv, see Pluckthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, N.Y., pp. 269 315 (1994).

The term “diabodies” refers to a small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.Acad Sci. USA 90: 6444 6448 (1993).

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick, et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 106 (1991).

The generation and preparation of antibodies is via any known method.See, for example, Green, et al., Production of Polyclonal Antisera, in:Immunochemical Protocols (Manson, ed.), pages 1 5 (Humana Press);Coligan, et al., Production of Polyclonal Antiscra in Rabbits, Rats Miceand Hamsters, in: Current Protocols in Immunology, section 2.4.1 (1992),which are hereby incorporated by reference for the generation andpreparation of polyclonal antibodies; Kohler & Milstein, Nature, 256:495(1975); Coligan, et al., sections 2.5.1 2.6.7; and Harlow, et al., in:Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub.(1988)), which are hereby incorporated by reference for the generationand preparation of monoclonal antibodies. Methods of generating antibodyfragments can be generated similar to the protocols described in forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, (1988).

The antibodies are attached on the linker by any known method. Theantibody can be attached directly to a selected functional group on thelinker in some embodiments. Alternatively, the antibodies can be linkedindirectly to the linker via a second linker or spacer in otherembodiments.

V. Detectable Analytes/Biomarkers and Disease States/Uses

In one aspect, the target analyte is a protein. There are a large numberof possible proteinaceous target analytes that may be detected using thepresent embodiments herein. By “proteins” or grammatical equivalentsherein is meant proteins, oligopeptides and peptides, derivatives andanalogs, including proteins containing non-naturally occurring aminoacids and amino acid analogs, and peptidomimetic structures. The sidechains may be in either the (R) or the (S) configuration. In someembodiments, the amino acids are in the (S) or L-configuration.

Suitable protein analytes include, but are not limited to, (1)immunoglobulins, particularly IgEs, IgGs and IgMs, and particularlytherapeutically or diagnostically relevant antibodies, including but notlimited to, for example, antibodies to human albumin, apolipoproteins(including apolipoprotein E), human chorionic gonadotropin, cortisol,α-fetoprotein, thyroxin, thyroid stimulating hormone (TSH),antithrombin, antibodies to pharmaceuticals (including antieptilepticdrugs (phenytoin, primidone, carbariezepin, ethosuximide, valproic acid,and phenobarbitol), cardioactive drugs (digoxin, lidocaine,procainamide, and disopyramide), bronchodilators (theophylline),antibiotics (chloramphenicol, sulfonamides), antidepressants,immunosuppresants, abused drugs (amphetamine, methamphetamine,cannabinoids, cocaine and opiates) and antibodies to any number ofviruses (including orthomyxoviruses, (e.g., influenza virus),paramyxoviruses (e.g., respiratory syncytial virus, mumps virus, measlesvirus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. m rubella virus), parvoviruses, poxviruses (e.g.,variola virus, vaccinia virus), enteroviruses (e.g., poliovirus,coxsackievirus), hepatitis viruses (including A, B and C), herpesviruses(e.g., Herpes simplex virus, varicella-zoster virus, cytomegalovirus,Epstein-Barr virus), rotaviruses, Norwalk viruses, hantavirus,arenavirus, rhabdovirus (e.g., rabies virus), retroviruses (includingHIV, HTLV-I and -II), papovaviruses (e.g., papillomavirus),polyomaviruses, and picornaviruses, and the like), and bacteria(including a wide variety of pathogenic and non-pathogenic prokaryotesof interest including Bacillus; Vibrio, e.g. V. cholerae; Escherichia,e.g. Enterotoxigenic E. coli, Shigella, e.g., S. dysenteriae;Salmonella, e.g., S. typhi, Mycobacterium e.g. M. tuberculosis, M.leprae; Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C.perfringens; Cornyebacterium, e.g. C. diphtherias; Streptococcus, S.pyogenes, S. pneumoniae, Staphylococcus, e.g., S. aureus; Haemophilus,e.g. H. influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae;Yersinia, e.g., G. lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa,P. putida; Chlamydia, e.g. C. trachomatis; Bordetella, e.g., B.pertussis; Treponema, e.g. T palladium; and the like); (2) enzymes (andother proteins), including but not limited to, enzymes used asindicators of or treatment for heart disease, including creatine kinase,lactate dehydrogenase, aspartate amino transferase, troponin T,myoglobin, fibrinogen, cholesterol, triglycerides, thrombin, tissueplasminogen activator (tPA); pancreatic disease indicators includingamylase, lipase, chymotrypsin and trypsin; liver function enzymes andproteins including cholinesterase, bilirubin, and alkaline phosphotase;aldolase, prostatic acid phosphatase, terminal deoxynucleotidyltransferase, and bacterial and viral enzymes such as HIV protease; (3)hormones and cytokines (many of which serve as ligands for cellularreceptors) such as erythropoietin (EPO), thrombopoietin (TPO), theinterleukins (including IL-1 through IL-17), insulin, insulin-likegrowth factors (including IGF-1 and -2), epidermal growth factor (EGF),transforming growth factors (including TGF-α and TGF-β), human growthhormone, transferrin, epidermal growth factor (EGF), low densitylipoprotein, high density lipoprotein, leptin, VEGF, PDGF, ciliaryneurotrophic factor, prolactin, adrenocorticotropic hormone (ACTH),calcitonin, human chorionic gonadotropin, cotrisol, estradiol, folliclestimulating hormone (FSH), thyroid-stimulating hormone (TSH), leutinzinghormone (LH), progeterone, testosterone, and (4) other proteins(including α-fetoprotein, carcinoembryonic antigen CEA).

In some embodiments, a protein analyte is a biomarker for a disease,disorder or condition. Exemplary biomarkers include, but are not limitedto, (e.g., PSA, BRCA1, BRCA1, Her2/neu, AFP (α-feto protein), B2M. (β-2microglobulin), BTA (Bladder tumor antigen), CA 15-3 (Cancer antigen15-3), CA 19-9 (Cancer antigen 19-9), hCG (Human chorionicgonadotropin), CA 72-4 (Cancer antigen 72-4), CA-125 (Cancer antigen125), Calcitonin, CEA (Carcino-embryonic antigen), EGFR (Her-1),Estrogen receptors, Progesterone receptors, Monoclonal immunoglobulins,NSE (Neuron-specific enolase), NMP22, thyroglobulin, monoclonalimmunoglobulins, NSE (Neuron-specific enolase), progesterone receptorsPSA (Prostate specific antigen), total and free, prostate-specificmembrane antigen (PSMA), prostatic acid phosphatase (PAP), S-100, andTA-90, or a portion or variation or fragment thereof. In certaininstances, the biomarker is a biomarker for cancer. In other instances,the biomarker is a biomarker for bacterial infections. In furtherinstances, the biomarker is a biomarker for Staphylococcus orStreptococcus bacterial infections.

VI. Samples

Samples for the detection systems and methods described herein can beany material suspected of containing an analyte. In some embodiments,the sample can be any source of biological material which comprisesproteins that can be obtained from a living organism directly orindirectly, including cells, tissue or fluid, and the deposits left bythat organism, including viruses, mycoplasma, and fossils. Typically,the sample is obtained as or dispersed in a predominantly aqueousmedium. Nonlimiting examples of the sample include blood, urine, semen,milk, sputum, mucus, a buccal swab, a vaginal swab, a rectal swab, anaspirate, a needle biopsy, a section of tissue obtained for example bysurgery or autopsy, plasma, serum, spinal fluid, lymph fluid, theexternal secretions of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, tumors, organs, samples of in vitrocell culture constituents (including but not limited to conditionedmedium resulting from the growth of cells in cell culture medium,putatively virally infected cells, recombinant cells, and cellcomponents), and a recombinant library comprising proteins, peptides,and the like.

The sample can be a positive control sample which is known to contain atarget analyte. A negative control sample can also be used which,although not expected to contain the analyte, is suspected of containingit (via contamination of one or more of the reagents) or anothercomponent capable of producing a false positive, and is tested in orderto confirm the lack of contamination by the target analyte of thereagents used in a given assay, as well as to determine whether a givenset of assay conditions produces false positives (a positive signal evenin the absence of target analyte in the sample).

The sample can be diluted, dissolved, suspended, extracted or otherwisetreated to solubilize and/or purify any target analyte present or torender it accessible to reagents which are used in an amplificationscheme or to detection reagents. Where the sample contains cells, thecells can be lysed or permeabilized to release the polynucleotideswithin the cells. One step permeabilization buffers can be used to lysecells which allow further steps to be performed directly after lysis,for example a polymerase chain reaction.

VII. Devices for Use, Three Electrode Systems, Scanning Methods

Electron transfer is generally initiated electronically, with theapplication of at least a first electric potential applied to the systemcomprising the electrode and redox reporter. Precise control andvariations in the applied potential can be via a potentiostat and eithera three electrode system (one reference, one sample (or working) and onecounter electrode) or a two electrode system (one sample and one counterelectrode). In some embodiments, a potentiostat with a three electrodesystem is employed with a Ag/AgCl reference electrode and a platinumwire auxiliary electrode. Electrical signals can be measured either bycyclic voltammetry or differential pulse voltammetry. In certaininstances, electrical signals are measured by cyclic voltammetry at ascan rate of about 50 m/v, of about 80 m/v, of about 100 m/v, of about120 m/v, or of about 150 m/v. In other instances, electrical signals arcmeasured by differential pulse voltammetry with a potential step ofabout 1-5 mV or about 2-10 mV, pulse amplitude of about 25-50 mV orabout 40-75 mV, pulse about 25-50 ms or about 40-75 ms, and pulse periodof about 10-100 ms or about 25-150 ms or about 50-200 ms.

In other instances, other means of detecting electrochemical potentials,including but not limited to potentiometric, amperometric, pulsevoltammetry, cyclic voltammetry, broadband frequency response,impedance, or other electrochemical methods may be used to transduceoutput signals from the electrochemically modified electrodes of thesystems and methods described herein.

VIII. Kits

Kits comprising components for the described systems and for performingthe described methods are also provided. In some embodiments, a kitcomprises one or more of the following components including anelectrode, reagents to form a linker on the surface of the electrode,one or more antibodies and a redox reporter. The components of a kit canbe retained by a housing. Instructions for using the kit to perform adescribed method can be provided with the housing, and can be providedin any fixed medium. The instructions may be located inside the housingor outside the housing, and may be printed on the interior or exteriorof any surface forming the housing that renders the instructionslegible. A kit may be in multiplex form for detection of one or moredifferent target analytes.

EXAMPLES Example 1: Preparation of Electrodes and Linkers

Chips are cleaned by sonication in acetone for 5 min, rinsed withisopropyl alcohol and DI water for 30 s, and dried with a flow of air.Electrodeposition is performed at room temperature; 5 μm apertures onthe fabricated electrodes arc used as the working electrode and arecontacted using the exposed bond pads. Gold (Au) sensors are made usinga deposition solution containing 20 mM solution of HAuCl₄ and 0.5 M.HCl. The Au sensors are formed using DC potential amperometry at about0-250 mV for about 100-300 s. Alternatively, Au structures may also beformed using chronopotentiometry, for example at about 15-40 nA forabout 25-60 s.

Example 2: Coupling of Antibodies to Linkers

An aqueous solution containing 1-50 mM of 4-aminothiophenol or mercaptopropionic acid is applied for 10-20 h at room temperature on AU sensors.The sensors are then washed with DI water 2-3 times for 2-4 minutes. Thetreated sensors were allowed to react at room temperature with 1.5-3.0%glutaraldehyde in water for 1 h followed by washing with DI water 2-3times for 2-4 minutes. The functionalized sensors are then reacted withPBS containing 5-25 m/ml antibody at room temperature for 1-2 h. Thesensors are washed with PBS 2-3 times for 3-6 minutes. The unreactedaldehyde groups are blocked with PBS containing 1% (W/V) bovine serumalbumin (BSA) for 1-2 hours at room temperature. The sensors are thenwashed 3-4 times with PBS for 4-7 minutes.

Example 3: Detection of Cancer Biomarker, CA-125

The development of platforms for the sensitive and straightforwardmeasurement of protein levels in clinical samples is an important goalthat will facilitate expanded use of protein biomarkers in diseasediagnosis. In order to provide useful information, detection schemesmust exhibit high levels of specificity, low limits of detection, androbust performance in biological fluids like blood and serum. Given theemergence of multi-protein signatures for cancer and other diseases,multiplexing is also a valuable feature. The inclusion of internal andexternal controls and calibrators also requires multiplexing.

CA-125 is an epithelial antigen that has been used as a marker for thedetection of ovarian cancer. Several assays have been developed todetect CA-125, but most are not ideal either due to lack of sensitivityor the complexity of the detection procedure. The commercially availableCA-125 immunoassay has a detection limit of 15 U/ml, which is sufficientto detect levels of CA-125 that correlate with the presence of disease(35 U/ml), but does not allow the relevance of significantly lowerlevels to be studied accurately.

The protein detection system disclosed herein was adapted toelectrochemically detect a CA-125 cancer biomarker commonly present inovarian cancers, via a microelectrode sensor on-a-chip design. Thesensor chips allow for comparison of electrode sensors of differentsizes, as well as determining a limit of detection exhibited down to 0.1U/ml. The readout was performed in a single step involving theintroduction of a non-covalently attached redox reporter group. Thedetection system reported exhibited was specific, with analysis ofCA-125 in human serum. The multiplexing of the system allowed theanalysis of the biomarker to be performed in parallel with an abundantserum protein, human serum albumin (HSA), for internal calibration.

Materials. CA-125 antigen and human serum from AB donors, anti humanserum albumin (HSA) antibody, HAuCl₄ solution, potassium ferricyanide(K3[Fc(CN)₆]), potassium ferrocyanide trihydrade (K2[Fe(CN)_(6.3)H₂O),50% (w/w) glutaraldehyde, and cystamine were purchased from SigmaAldrich. Anti CA-125 antibody was obtained from KalGene PharmaceuticalsInc., Canada. ACS-grade acetone and isopropyl alcohol (IPA) wereobtained from EMD (USA); 6 N hydrochloric acid was purchased from VWR(USA). Phosphate-buffered saline (PBS, pH 7.4, 1×) was obtained fromTrivitrogen. Human whole blood was obtained from Bioreclaimation(Westbury, N.Y.).

Chip & Electrode Fabrication: Chips were fabricated at the CanadianPhotonics Fabrication Center. Briefly, three inch silicon wafers werepassivated using a thick layer of thermally grown silicon dioxide. A 350nm gold layer was deposited on the chip using electron-beam-assistedgold evaporation. The gold film was patterned using standardphotolithography and a lift-off process. A 500 nm layer of insulatingsilicon dioxide was deposited using chemical vapor deposition; 5 μmapertures were imprinted on the electrodes using standardphotolithography, and 2 mm×2 mm bond pads were exposed using standardphotolithography. FIG. 1a depicts a photograph of an exemplary sensorchip (left) and representative layers (right).

Chips were cleaned by sonication in acetone for 5 min, rinsed withisopropyl alcohol and DI water for 30 s, and dried with a flow of air.Electrodeposition was performed at room temperature; 5 μm apertures onthe fabricated electrode sensors were used as the working electrode andwere contacted using the exposed bond pads. Three different goldelectrode sensors were made using a deposition solution containing 20 mMsolution of HAuCl₄ and 0.5 M HCl using a process similar to theprocedure as described in International Application Ser. No.PCT/CA2009/001212 (published as WO 2010/025547). The 100 micron and 30micron gold

electrode structures were formed using DC potential amperometry at 0 mVfor 200 s and 150 mV for 200 s respectively; and 8 micron gold electrodestructures were formed using chronopotentiometry at 30 nA for 50 s.FIGS. 2a, 2c and 2e depict SEM images of the 100 micron, 30 micron and 8micron gold electrode structures respectively.

Determination of surface area of the sensors. The surface area of Ausensors was calculated by integrating the Au oxide reduction peak areaobtained from cyclic voltammogram in the 50 mM H₂SO₄. In the forwardscan, a monolayer of chemisorbed oxygen is formed and then it is reducedin the reverse scan. The reduction charge per microscopic unit area hasbeen experimentally determined as 500 μC/geometric cm². The surface areawas calculated by integrating the reduction peak (ca. 0.812 V vs.Ag/AgCl) to obtain the reduction charge, and dividing this by 500μC/geometric cm².

Antibody Modification of the Electrodes: An aqueous solution containing10 mM cystamine was applied for 16 h at room temperature on goldelectrode sensors in order to form a uniform linker on the surface ofthe electrodes. Then, the electrodes were washed with DI water twice for2 minutes. The linker was allowed to react at room temperature with 2.5%glutaraldehyde in water for 1 h followed by washing with DI water twicefor 2 minutes. The functionalized electrodes were then reacted with PBScontaining 10 μg/ml anti CA-125 antibody or anti Human Serum Albumin(HSA) antibody at room temperature for 1 h. The electrodes were washedwith PBS twice for 5 minutes. The unreacted aldehyde groups were blockedwith PBS containing 1% (W/V) bovine serum albumin (BSA). The electrodeswere then washed three times with PBS for 5 min Solutions containingdifferent concentrations of CA-125 in PBS or serum were applied to theantibody-modified electrode for 40 min at 37° C. The electrodes werewashed with PBS prior to electrochemical readout.

Electrochemical Analysis and Scanning Electron Microscopy (SEM):Electrochemical experiments were carried out using a BioanalyticalSystems Epsilon potentiostat with a three-electrode system featuring aAg/AgCl reference electrode and a platinum wire auxiliary electrode.Electrochemical signals were measured in a 10 mM phosphate buffersolution (pH 7) containing 2.5 mM K3[Fe(CN)_(6], 2.5) mM K2[Fe(CN)₆],and 0.1 M. KCl. Cyclic voltammetry (CV) was obtained with a scan rate of100 mV/s and differential pulse voltammetry (DPV) signals were obtainedwith a potential step of 5 mV, pulse amplitude of 50 mV, pulse with 50ms, and a pulse period of 100 ms. Signal changes corresponding to targetprotein binding to the antibody were calculated as follows: ΔI %={(meanI₀)−(mean I_(c))}/mean I₀×100 (where mean I₀=mean current at zero targetconcentration, mean I_(c)=mean current at any concentration of target).The SEM images were obtained using a Hitachi S-3400 SEM.

Three different structures were fabricated on chips using differentelectrochemical methods and conditions. 100 micron (FIG. 2a ), 30 micron(FIG. 2c ), and 8 micron (FIG. 2e ) sensors were generated by varyingthe electrodeposition conditions. The surface areas of the sensors weremeasured by scanning in sulfuric acid and measuring the amount of oxideformed and stripped from the surface, and areas of approximately 4×10⁻⁶cm²(8 micron sensor), 3×10⁻⁵ cm² (30 micron sensor, and 1×10⁻⁴ cm² (100micron sensor) were obtained. The capacitive currents measured in buffersolution (FIG. 2g ) were consistent with these values. Before testingthe sensors for protein detection, cyclic voltammograms were obtainedfor each structure in a solution containing ferrocyanide andferricyanide (FIGS. 2b, 2d, and 2f ). The 100 micron sensor displayeddiffusion-limited currents and the 8 and 30 micron sensors exhibitedplateau currents consistent with those expected for microelectrodes.

The detection limits of the immunosensors formed on the threedifferently-sized sensors were evaluated by measuring differential pulsevoltammograms (DPVs) of [Fe(CN)6]_(3-/4-) solutions before and afterincubation with CA-125 for 40 minutes. With the largest sensors (FIG. 3a), concentrations of 10 U/ml were required for appreciable signalchanges. With the intermediate 30 micron sensors, the detection limitapproached 1 U/ml (FIG. 3b ). With the smallest, 8 micron sensors, 0.1U/ml of CA-125 was detectable (FIG. 3c ). 0.1 U/ml of CA-125 antigen isequivalent to ˜500 fM of CA-125 or 100 pg/ml.

To evaluate the exemplary detection system with biological fluids,detection of CA-125 samples in human serum was performed. Serum is ahighly complex biological fluid containing large amounts of proteins andother molecules. Human serum albumin (HSA) was chosen as an internalstandard due to constant and high concentrations of HSA in human sera.On a multiplex sensor chip, immunosensors for CA-125 and HSA weredeveloped by forming layer of anti CA-125 antibody and anti HSA antibodyrespectively (FIG. 1a ). Human serum spiked with differentconcentrations of CA-125 was applied to the multiplexed immunosensor andDPVs were recorded in a mixed solution of ferrocyanide and ferricyanide.FIG. 5a shows the AI % (with respect to serum with 0 U/ml CA-125) valuesobtained for human serum spiked with different concentrations of CA-125.AI % for sensors modified with anti CA-125 antibody increased withincreasing concentrations of CA-125, whereas the AI % for HSA wasessentially constant for the immunosensors modified with anti-HSAantibody. The detection of CA-125 in parallel with a serum protein is auseful means to provide absolute measurements of the cancer biomarker.

The performance of this sensing system was also investigated in whole,unprocessed blood. Blood samples were spiked with CA-125 and theanalysis was performed in the same manner as the serum and bufferstudies. Interestingly, the same limit of detection was obtained, with0.1 U/ml clearly resolved over background levels as depicted in FIG. 5b. This level of sensitivity achieved with an unprocessed blood sampleindicates that this simple, straightforward electrochemical proteindetection assay is also remarkably robust.

The following table depicts the approximate detection limit of CA-125for various electrode sizes:

Electrode diameter Approximate limit of detection  5 microns 0.1 U/ml 50 microns 1 U/ml 100 microns 10 U/ml 2000 microns (2 mm) 100 U/ml

REFERENCES

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While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for electrochemical detection of acancer antigen, comprising: providing a nanostructured microelectrodeincluding a linker on its surface, the linker attached to an antibody orfragment thereof and capable of binding the antigen; contacting thenanostructured microelectrode with a sample including the antigen andwith a redox reporter capable of electron transfer with themicroelectrode; binding of the antigen to the antibody or fragmentthereof, the binding hindering electron transfer between the redoxreporter and the nanostructured microelectrode, the nanostructuredmicroelectrode having a diameter of about 100 microns or less, thenanostructured microelectrode having a detection limit for the biomarkerfrom about 0.1 U/ml to about 10 U/ml; applying a potential to thenanostructured microelectrode; measuring, in response to the appliedpotential, an electrochemical signal generated by the nanostructuredmicroelectrode; comparing the electrochemical signal to a controlsignal; and deeming a change of the electrochemical signal detectedrelative to the control signal as indicative of the presence of thecancer antigen in the sample.
 2. The method of claim 1, wherein thecancer antigen is selected from the group consisting of bladder tumorantigen, cancer antigen 15-3, cancer antigen 19-9, cancer antigen 72-4,cancer antigen 125 (CA-125), carcino-embryonic antigen, prostatespecific antigen (PSA), prostate-specific membrane antigen, prostaticacid phosphatase, and TA-90, or a portion, variation or fragmentthereof.
 3. The method of claim 1, wherein the linker includes afunctional group capable of direct or indirect coupling to the antibodyor fragment thereof.
 4. The method of claim 1, wherein thenanostructured microelectrode is labeled with multiple copies of theantibody or fragment thereof.
 5. The method of claim 1, wherein thenanostructured microelectrode is capable of interfacial electrontransfer.
 6. The method of claim 1, wherein the measuring step includesemploying a voltammetry-based approach.
 7. The method of claim 1,wherein the nanostructured microelectrode is a first nanostructuredmicroelectrode, the linker is a first linker, the antibody or fragmentthereof is a first antibody or fragment thereof, the electrochemicalsignal is a first electrochemical signal, and the control signal is afirst control signal, the method further comprising: providing a secondnanostructured microelectrode including a second linker on its surface,the second linker attached to a second antibody or fragment thereof andcapable of binding a second antigen of the sample; contacting the secondnanostructured microelectrode with the sample and with the redoxreporter capable of electron transfer with the second microelectrode;binding of the second antigen to the second antibody or fragmentthereof, the second antigen being different than the first antigen;applying the potential to the second nanostructured microelectrode;measuring, in response to the applied potential, a secondelectrochemical signal generated by the second nanostructuredmicroelectrode; comparing the second electrochemical signal to a secondcontrol signal; and deeming a change of the second electrochemicalsignal detected relative to the second control signal as indicative ofthe presence of the second antigen in the sample.
 8. The method of claim7, wherein providing the first nanostructured electrode and providingthe second nanostructured electrode includes providing a microfabricatedchip having the first nanostructured electrode and the secondnanostructured electrode fabricated thereon.
 9. The method of claim 7,wherein the second nanostructured microelectrode is a reference controlto the first nanostructured microelectrode.
 10. The method of claim 7,wherein the first and second nanostructured microelectrode are ondifferent microfabricated chips, wherein providing the firstnanostructured electrode includes providing a first microfabricated chiphaving the first nanostructured electrode fabricated thereon, andwherein providing the second nanostructured electrode includes providinga second microfabricated chip having the second nanostructured electrodefabricated thereon.
 11. The method of claim 7, wherein the secondantigen includes abundant serum protein.
 12. The method of claim 7,wherein the first and second linkers each include a functional groupcapable of direct or indirect coupling to the antibody or fragmentthereof.
 13. The method of claim 7, wherein the first nanostructuredmicroelectrode is labeled with multiple copies of the first antibodyand/or the second nanostructured microelectrode is labeled with multiplecopies of the second antibody or fragment thereof.
 14. The method ofclaim 7, wherein the deeming includes deeming a reduction in a signalmagnitude of the electrochemical signal detected relative to the controlsignal as indicative of the presence of the antigen in the sample. 15.The method of claim 1, wherein the deeming includes deeming a reductionin a signal magnitude of the electrochemical signal detected relative tothe control signal as indicative of the presence of the antigen in thesample.
 16. The method of claim 1, wherein the antibody or fragmentthereof is selected from the group consisting of polyclonal antiserum,polyclonal antibody, monoclonal antibody, Fab fragment, Fab′ fragment,F(ab′)2 fragment, Fv fragment, single chain antibody, CDR peptide anddiabodies.
 17. A method for electrochemical detection of a viralantigen, comprising: providing a nanostructured microelectrode includinga linker on its surface, the linker attached to an antibody or fragmentthereof and capable of binding the antigen; contacting thenanostructured microelectrode with a sample including the antigen andwith a redox reporter capable of electron transfer with themicroelectrode; binding of the antigen to the antibody or fragmentthereof, the binding hindering electron transfer between the redoxreporter and the nanostructured microelectrode, the nanostructuredmicroelectrode having a diameter of about 100 microns or less, thenanostructured microelectrode having a detection limit for the antigenfrom about 0.1 U/ml to about 10 U/ml; applying a potential to thenanostructured microelectrode; measuring, in response to the appliedpotential, an electrochemical signal generated by the nanostructuredmicroelectrode; comparing the electrochemical signal to a controlsignal; and deeming a change of the electrochemical signal detectedrelative to the control signal as indicative of the presence of theviral antigen in the sample.
 18. The method of claim 17, wherein theviral antigen is associated with orthomyxoviruses, (e.g., influenzavirus), paramyxoviruses (e.g., respiratory syncytial virus, mumps virus,measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. m rubella virus), parvoviruses, poxviruses (e.g.,variola virus, vaccinia virus), enteroviruses (e.g., poliovirus,coxsackievirus), hepatitis viruses (including A, B and C), herpesviruses(e.g., Herpes simplex virus, varicella-zoster virus, cytomegalovirus,Epstein-Barr virus), rotaviruses, Norwalk viruses, hantavirus,arenavirus, rhabdovirus (e.g., rabies virus), retroviruses (includingHIV, HTLV-I and -II), papovaviruses (e.g., papillomavirus),polyomaviruses, or picornaviruses.
 19. A method for electrochemicaldetection of an antigen, comprising: providing a nanostructuredmicroelectrode including a cystamine, cysteamine, mercapto propionicacid or 4-aminothiophenol linker on its surface, the linker attached toan antibody or fragment thereof and capable of binding the antigen;contacting the nanostructured microelectrode with a sample including theantigen and with a redox reporter capable of electron transfer with themicroelectrode; binding of the antigen to the antibody or fragmentthereof, the binding hindering electron transfer between the redoxreporter and the nanostructured microelectrode, the nanostructuredmicroelectrode having a diameter of about 100 microns or less, thenanostructured microelectrode having a detection limit for the antigenfrom about 0.1 U/ml to about 10 U/ml; applying a potential to thenanostructured microelectrode; measuring, in response to the appliedpotential, an electrochemical signal generated by the nanostructuredmicroelectrode; comparing the electrochemical signal to a controlsignal; and deeming a change of the electrochemical signal detectedrelative to the control signal as indicative of the presence of theantigen in the sample.
 20. The method of claim 19, wherein the linker isattached to the antibody or fragment thereof via a second linker. 21.The method of claim 20, wherein the second linker is glutaraldehyde orformaldehyde.
 22. A method for electrochemical detection of a cancerbiomarker, comprising: providing a nanostructured microelectrodeincluding a linker on its surface, the linker attached to an antibody orfragment thereof and capable of binding the biomarker; contacting thenanostructured microelectrode with a sample including the biomarker andwith a redox reporter capable of electron transfer with themicroelectrode; binding of the biomarker to the antibody or fragmentthereof, the binding hindering electron transfer between the redoxreporter and the nanostructured microelectrode, the nanostructuredmicroelectrode having a diameter of about 100 microns or less, thenanostructured microelectrode having a detection limit for the biomarkerfrom about 0.1 U/ml to about 10 U/ml; applying a potential to thenanostructured microelectrode; measuring, in response to the appliedpotential, an electrochemical signal generated by the nanostructuredmicroelectrode; comparing the electrochemical signal to a controlsignal; and deeming a change of the electrochemical signal detectedrelative to the control signal as indicative of the presence of thecancer biomarker in the sample.
 23. The method of claim 22, wherein thecancer biomarker is BRCA1, Her2/neu, alpha-feto protein, beta-2microglobulin, bladder tumor antigen, cancer antigen 15-3, cancerantigen 19-9, human chorionic gonadotropin, cancer antigen 72-4, cancerantigen 125 (CA-125), calcitonin, carcino-embryonic antigen, EGFR,Estrogen receptors, Progesterone receptors, Monoclonal immunoglobulins,neuron-specific enolase, NMP22, thyroglobulin, progesterone receptors,prostate specific antigen (PSA), prostate-specific membrane antigen,prostatic acid phosphatase, S-100, and TA-90, or a portion, variation orfragment thereof.
 24. A detection system for electrochemically detectingan antigen, comprising: (i) a nanostructured microelectrode including alinker on its surface, wherein the nanostructured microelectrode has adiameter of about 100 microns or less and a detection limit for theantigen from about 0.1 U/ml to about 10 U/ml, and wherein the linker isattached to an antibody or fragment thereof capable of binding theantigen; and (ii) a redox reporter.